Thrombin solution and methods of use thereof

ABSTRACT

Provided are methods for lyophilization of an aqueous thrombin solution, thrombin solutions for use in such lyophilization methods, and solid thrombin compositions produced by such methods.

FIELD OF THE INVENTION

The invention relates to the field of pharmaceutical solutions, and morespecifically to methods for lyophilization of an aqueous thrombinsolution, thrombin solutions for use in such lyophilization methods, andsolid thrombin compositions produced by such methods.

BACKGROUND OF THE INVENTION

Thrombin is a proteolytic enzyme formed from prothrombinin plasma duringthe clotting process.

Thrombin is widely used in clinical applications as a coagulation factorto staunch bleeding of wounds by conversion of fibrinogen to fibrin. Itis a common component of surgical dressings, and has been used incombination with fibrinogen and other coagulation proteins intwo-component hemostatic systems such as fibrin glues, adhesives, andsealants.

It is known to lyophilize a solution of thrombin to produce a solidthrombin composition prior to storage, in order to reduce proteindegradation, wherein the solid thrombin composition may be reconstitutedprior to use. Lyophilization typically refers to the process of freezinga mixture and then reducing the concentration of water e.g. bysublimation to levels which do not support biological or chemicalreactions. The porous and spongy solid material resulting from thelyophilization process is referred to as a cake. It is desirable forsuch solid thrombin compositions to have low water content (e.g. lessthan about 3%), to retain structural and functional stability for longperiods), preferably at room temperature, and to retain a highpercentage of thrombin activity upon reconstitution as compared to theactivity of thrombin in the solution prior to lyophilization.

Examples of background art thrombin solutions are described in EP813598B1; U.S. Pat. Nos. 5,605,884; 4,877,608; US 2010/0074865; U.S.Pat. No. 5,733,873; EP 1766003; and US 2010/0168018. The background artthrombin solutions vary considerably in the number and types ofexcipients present, and in the concentrations of the individualexcipients.

Cryoprotectants or stabilizers are commonly used in thrombin solutionsto protect the thrombin from denaturation or activity loss due tofreezing stress, to stabilize the protein in subsequent productionsteps, and to extend the shelf life. Examples of cryoprotectants includesaccharides such as sucrose, lactose, and trehalose; sugar alcohols suchas mannitol; and surfactants such as polyethylene glycol, TritonX-100,TWEEN-20, and TWEEN-80. Besides functioning as stabilizers, mannitol andsucrose (to a lesser degree) are also used as bulking agents which helpto provide a cake having a strong physical structure. Use of bulkingagents is especially important for formulation with a low solid materialcontent (per volume).

Human Serum Albumin (HSA) is also widely used in biopharmaceuticalformulations as a stabilizing and bulking agent (H. R. Constantino, M.J. Pikal: Lyophilization of Biopharmaceuticals, Springer, 2004).

EP 813598B1 discloses a simple thrombin formulation for lyophilization,containing 40 mM gluconic acid, 20 mM tri-sodium citrate and 150 mMNaCl; U.S. Pat. Nos. 5,605,884 and 4,877,608 disclose formulationscontaining up to 10% saccharides such as sucrose, mannitol or maltose;US 2010/0074865 discloses formulation containing 5.7% lactose, 3.1%trehalose and 0.001% TWEEN-80; and U.S. Pat. No. 5,733,873 disclosesformulations including 0.001-0.025% polysorbate 80 (TWEEN-80) with orwithout 0.1% PEG 4000 and 2% mannitol.

Sodium chloride (NaCl) is commonly used for reducing proteinprecipitation and aggregation during lyophilization of biopharmaceuticalformulations. However, EP 1766003 discloses that NaCl can be problematicbecause it lowers the glass transition temperature, therebynecessitating a low temperature of primary drying and a prolonged dryingcycle time. Also, US 2010/0168018 discloses a formulation without NaCl,or with NaCl present in trace amounts.

Methods of lyophilization of such known thrombin solutions arefrequently of a very long duration, which increases the cost of theprocess and/or result in solid thrombin compositions having a relativelyhigh water content and/or which retain a relatively low percentage ofthrombin activity upon reconstitution, as compared to the thrombinactivity in the solution prior to lyophilization.

SUMMARY OF THE INVENTION

The invention, in some embodiments thereof, relates to methods forlyophilization of an aqueous thrombin solution, thrombin solutions foruse in such lyophilization methods, and solid thrombin compositionsproduced by such methods. In some embodiments, the solutions and methodsdisclosed herein enable solid thrombin compositions to be prepared usinglyophilization methods which are shorter than known methods forlyophilization of thrombin solutions, for example, 37% shorter, therebyincreasing lyophilization capacity and cost-effectiveness.

In some embodiments, the lyophilization methods described herein resultin solid thrombin compositions having a relatively low water contentand/or which are highly stable at room temperature over relatively longperiods of time, and/or which exhibit a high thrombin activity recoveryupon reconstitution. In some embodiments, the method comprises use of anoptimal aqueous thrombin formulation.

Aspects and embodiments of the invention are described in thespecification hereinbelow and in the appended claims.

According to an aspect of some embodiments described herein, there isprovided a method for lyophilization of an aqueous thrombin solution,the method comprising providing the aqueous thrombin solution comprisingabout 1 to less than about 4.6% (w/v) saccharide or sugar alcohol; atleast about 0.7 to lower than about 1.75% (w/v) sodium chloride; about0.2 to about 3% albumin; calcium chloride; and sodium acetate; andlyophilizing the aqueous thrombin solution.

In some embodiments, the presence of calcium in the aqueous thrombinsolution stabilizes the thrombin structure thereby preserving itsactivity during lyophilization. Also, calcium is required to supportthrombin-hemostatic activity. Following lyophilization of an aqueousthrombin solution, a porous and spongy solid material, referred to as acake (also referred to herein as a solid thrombin composition) isobtained.

In some embodiments, the aqueous thrombin solution comprises about 200to about 2000 IU/ml thrombin; about 0.3 to about 1.5% (w/v) calciumchloride; and about 0.14 to about 1% (w/v) sodium acetate.

In some embodiments, the aqueous thrombin solution comprises about 1.6to less than about 4.6% (w/v) saccharide or sugar alcohol; about 0.7 toabout 1.7% (w/v) sodium chloride; higher than about 0.2 to lower thanabout 3% albumin; about 0.3 to about 1.2% (w/v) calcium chloride; andabout 0.14 to about 0.7% (w/v) sodium acetate.

In some embodiments, the saccharide or sugar alcohol is present at aconcentration of 2% (w/v); albumin is present at a concentration ofabout 0.6% (w/v); sodium chloride is present at a concentration of about0.76% (w/v); calcium chloride is present at a concentration of about0.6% (w/v) and sodium acetate is present at a concentration of about0.27% (w/v).

In some embodiments, the aqueous thrombin solution consists essentiallyof thrombin; saccharide or sugar alcohol; sodium chloride; albumin;calcium chloride; and sodium acetate.

In some embodiments, the saccharide comprises a monosaccharide(optionally selected from the group consisting of glucose, fructose,galactose, xylose and ribose) and/or disaccharide (optionally selectedfrom the group consisting of sucrose, maltose and lactose). In someembodiments, the disaccharide comprises sucrose and/or maltose,optionally at a concentration of about 2% (w/v).

In some embodiments, the sugar alcohol comprises amonosaccharide-derived sugar alcohol, optionally selected from the groupconsisting of mannitol, sorbitol, and xylitol.

In some embodiments, the monosaccharide-derived sugar comprisesmannitol, optionally at a concentration of about 2% (w/v).

In some embodiments, the sugar alcohol is a disaccharide-derived sugaralcohol, optionally selected from the group consisting of maltitol,isomalt, and lactitol.

In some embodiments, the aqueous thrombin solution comprises a singlesaccharide (either monosaccharide or disaccharide) or sugar alcohol. Insome embodiments, the aqueous thrombin solution is devoid of more thanone kind of saccharide or sugar alcohol. In some embodiments, theaqueous thrombin solution is devoid of at least one of polyethyleneglycol and histidine.

In some embodiments, the method further comprises, prior tolyophilization, adjusting the pH of the aqueous thrombin solution to apH in the range of about 5.5 to about 9.

In some embodiments, the height of the aqueous thrombin solution in alyophilization vessel, as measured from the lowest point of the vessel,is no greater than about 10 mm, such as, for example, about 10 mm, about9 mm, about 8 mm, about 7 mm, or about 6 mm. In some exemplaryembodiments, the height is about 8 mm.

In some such embodiments, wherein the height of the aqueous thrombinsolution is no greater than about 10 mm, the total lyophilization timeis no greater than about 35 hours, such as for example, about 35 hours,about 34 hours, about 33 hours, about 32 hours, about 31 hours, about 30hours, or about 29 hours. In some exemplary embodiments, the totallyophilization time is no greater than about 30 hours.

In some embodiments, the height of the aqueous thrombin solution in alyophilization vessel is no greater than about 20 mm. In someembodiments, the height of the aqueous thrombin solution is in the rangeof about 15 to about 19 mm, such as, for example, 15 mm, 16 mm, 17 mm,18 mm, or 19 mm. In some exemplary embodiments, the height of theaqueous thrombin solution in a lyophilization vessel is about 17 mm.

In some such embodiments, wherein the height of the aqueous thrombinsolution is no greater than about 20 mm, the total lyophilization timeis no greater than about 68 hours.

In some embodiments, wherein the height of the aqueous thrombin solutionis no greater than about 20 mm, the total lyophilization time is shorterthan that of a lyophilization cycle using a control solution ofidentical height in a lyophilization vessel. A “control solution” has acomposition which is different than the composition described herein.The control composition can be different from the composition of theinvention by comprising additional or other types of excipients and/orin the concentrations of the individual excipients.

In some embodiments, the total lyophilization time as compared to thatusing known prior art solutions, is reduced by about 37%.

In some embodiments, lyophilizing comprises: i) subjecting the aqueousthrombin solution to a freezing procedure to produce a frozen thrombinsolution; ii) subjecting the frozen thrombin solution of step i) to aprimary drying procedure; and iii) subjecting the product of step ii) toa secondary drying procedure.

In some embodiments, the freezing procedure is carried out at a freezingtemperature of about −45° C. to about −55° C. In some embodiments,lypophilization is carried out on a lyophilizer shelf and the freezingprocedure comprises maintaining the lyophilizer shelf at a freezingtemperature (also referred to herein as a freezing soak) of about −45°C. to about −55° C., such as, for example, about −50° C.

In some embodiments, the freezing procedure is carried out at a pressureof about 1 atmosphere.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 10 mm (such as about8 mm), the freezing temperature is maintained for no longer than about 5hours.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 20 mm (such as 15-19mm, for example, about 17 mm), the freezing temperature is maintainedfor no longer than about 6 hours.

In some embodiments, the freezing temperature, such as the temperatureof the lyophilizer shelf, is reached over a period (also referred toherein as a freezing ramp) of no greater than about 2.5 hours.

In some embodiments, the primary drying procedure is carried out atabout −12° C. to about −18° C. In some embodiments, the primary dryingprocedure comprises maintaining the lyophilizer shelf at a primarydrying temperature (also referred to herein as a primary drying soak) ofabout −12° C. to about −18° C., such as about −12° C., about −13° C.,about −14° C., about −15° C., about −16° C., about −17° C., or about−18° C., at a pressure of about 100 μBar to about 160 μBar. In someexemplary embodiments, the primary drying temperature is about −15° C.

In some embodiments, the primary drying temperature, such as thetemperature of the lyophilizer shelf, is reached over a period (alsoreferred to herein as a primary drying ramp) of about 80 to about 90minutes.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 10 mm (such as, forexample, about 8 mm), the primary drying temperature and pressure aremaintained for no longer than about 13 hours, such as, for example,about 13 hours, about 12 hours, or about 11 hours.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 20 mm (such as, forexample, about 15 to about 19 mm, or about 17 mm), the primary dryingtemperature (freezing soak) and pressure are maintained for no longerthan about 31 hours.

In some embodiments, the secondary drying procedure is carried out atabout 20° C. to about 30° C. In some embodiments, the secondary dryingprocedure comprises maintaining the lyophilizer shelf at a secondarydrying temperature (also referred to herein as a secondary drying soak)of about 20° C. to about 30° C. (such as, for example, about 25° C.) anda pressure of less than about 50 μBar, such as, for example, less thanabout 20 μBar.

In some embodiments, the temperature of the secondary drying temperatureis reached over a period (also referred to as a secondary drying ramp)of about 60 to about 90 minutes.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 10 mm (such as, forexample, about 8 mm), the secondary drying temperature and pressure aremaintained for no longer than about 11 hours (such as, for example,about 9.5 to about 11 hours.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 20 mm (such as about15 to about 19 mm, for example about 17 mm), the secondary dryingtemperature and pressure are maintained for no longer than about 15hours. In some such embodiments, the method further comprises, prior tothe secondary drying procedure, an intermediate drying procedure,carried out at about 5 to about 15° C. In some embodiments, thesecondary drying procedure comprises maintaining the lyophilizer shelfat an intermediate drying temperature (also referred to herein as anintermediate drying soak) of about 5 to about 15° C., such as, forexample about 10° C. In some such embodiments, the pressure during theintermediate drying procedure is about 120 μBar. In some suchembodiments, the intermediate drying temperature is maintained for nolonger than about 13 hours.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 10 mm (such as, forexample, about 8 mm), lyophilization on a lyophilizer shelf comprises:

-   -   a) bringing the temperature of the lyophilizer shelf to a        freezing temperature of about −50° C., over a time period in the        range of about 1.5 to about 2.5 hours;    -   b) maintaining the freezing temperature for a time period in the        range of about 4 to about 6 hours;    -   c) increasing the temperature of the lyophilizer shelf of        step b) to a primary drying temperature of about −15° C. and        bringing the pressure to from about 100 to about 160 μBar, over        a time period in the range of about 50 to about 90 minutes;    -   d) maintaining the primary drying temperature and pressure for a        time period in the range of about 11 to about 13 hours;    -   e) increasing the temperature of the lyophilizer shelf of        step d) to a secondary drying temperature of about 25° C. and        decreasing the pressure to less than about 50 μBar over a time        period in the range of about 60 to about 90 minutes; and    -   f) maintaining the secondary drying temperature and pressure of        step e) for a time period in the range of about 9.5 to about 11        hours.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 20 mm (such as,about 15-19 mm, for example 17 mm), lyophilization on a lyophilizershelf comprises:

-   -   a) bringing the temperature of the lyophilizer shelf to a        freezing temperature of about −50° C., over a time period in the        range of about 1.5 to about 2.5 hours;    -   b) maintaining the freezing temperature of step a) for a time        period in the range of about 4 to about 6 hours to produce a        frozen thrombin solution;    -   c) increasing the temperature of the lyophilizer shelf of        step b) to a primary drying temperature of about −15° C. and        bringing the pressure to from about 100 to about 160 μBar, over        a time period in the range of about 30 to about 70 minutes;    -   d) maintaining the primary drying temperature and pressure for a        time period in the range of about 30 to about 32 hours;    -   e) increasing the temperature of the lyophilizer shelf of        step d) to a secondary drying temperature of about 25° C. and        decreasing the pressure to less than about 50 μBar, over a time        period in the range of about 40 to about 80 minutes; and    -   f) maintaining the secondary drying temperature and pressure for        a time period in the range of about 13 to about 17 hours.

In some embodiments, steps a) and b) are carried out at atmosphericpressure.

In some embodiments, there is provided a solid thrombin compositionobtainable according to any of the methods described herein.

In some embodiments, the solid thrombin composition has a water contentof not more than about 3% (w/w) and a thrombin activity recovery of atleast 95%.

In some embodiments, the solid thrombin composition has a water contentof not more than about 1.5% (w/w) and a thrombin activity recovery of atleast 98%.

In some embodiments, the solid thrombin composition is stable for atleast 2 years under non-freezing storage conditions, such as at roomtemperature.

In some embodiments, the solid thrombin composition comprises about19.5% to about 78% (w/w of total composition) mannitol, about 1% toabout 20% (w/w of total composition) sodium acetate; about 2% to about53% (w/w of total composition) albumin; about 2.5% to about 31% (w/w oftotal composition) calcium chloride; and about 6% to about 45% (w/wtotal composition) sodium chloride.

In some embodiments, the solid thrombin composition comprises about 22%to about 66% (w/w of total composition) mannitol, about 1.5% to about10% (w/w of total composition) sodium acetate; about 2.5% to about 43%(w/w of total composition) albumin; about 4% to about 17% (w/w of totalcomposition) calcium chloride; and about 9.5% to about 25% (w/w of totalcomposition) sodium chloride.

According to an aspect of some embodiments described herein, there isprovided an aqueous thrombin solution comprising about 1 to less thanabout 4.6% (w/v) saccharide or sugar alcohol; at least about 0.7 tolower than about 1.75% (w/v) sodium chloride; about 0.2 to about 3%(w/v) albumin; calcium chloride; and sodium acetate.

In some embodiments, the aqueous thrombin solution comprises about 200to about 2000 IU/ml thrombin; about 0.3 to about 1.5% (w/v) calciumchloride; and about 0.14 to about 1% (w/v) sodium acetate.

In some embodiments, the aqueous thrombin solution comprises about 1.6to less than about 4.6% (w/v) saccharide or sugar alcohol; about 0.7 toabout 1.7% (w/v) sodium chloride; higher than about 0.2 to lower thanabout 3% albumin; about 0.3 to about 1.2% (w/v) calcium chloride; andabout 0.14 to about 0.7% (w/v) sodium acetate.

In some embodiments of the aqueous thrombin solution, the saccharide orsugar alcohol is present at a concentration of about 2% (w/v); thealbumin is present at a concentration of about 0.6% (w/v), the sodiumchloride is present at a concentration of about 0.76% (w/v), the calciumchloride is present at a concentration of about 0.6% (w/v) and thesodium acetate is present at a concentration of about 0.27% (w/v).

In some embodiments, the aqueous thrombin solution consists essentiallyof thrombin; saccharide or sugar alcohol; sodium chloride; albumin;calcium chloride; and sodium acetate.

In some embodiments, the saccharide is a monosaccharide, such as amonosaccharide selected from the group consisting of glucose, fructose,galactose, xylose and ribose.

In some embodiments, the saccharide is a disaccharide, such as adisaccharide selected from the group consisting of sucrose, maltose andlactose.

In some embodiments, the sugar alcohol comprises amonosaccharide-derived sugar alcohol, such as a monosaccharide-derivedsugar alcohol is selected from the group consisting of mannitol,sorbitol, and xylitol. In some embodiments, the monosaccharide-derivedsugar alcohol comprises mannitol, optionally at a concentration of about2% (w/v).

In some embodiments, the aqueous thrombin solution comprises a singlesaccharide or sugar alcohol (such as one of sucrose alone, maltosealone, or mannitol).

In some embodiments, the aqueous thrombin solution is devoid of morethan one kind of saccharide or sugar alcohol.

In some embodiments, the aqueous thrombin solution is devoid of at leastone of polyethylene glycol and histidine.

In some embodiments, the aqueous thrombin has a pH in the range of about5.5 to about 9.

In some embodiments, there is provided a method for lyophilization of anaqueous thrombin solution, comprising providing an aqueous thrombinsolution as described herein; and lyophilizing the aqueous thrombinsolution.

In some embodiments, the height of the aqueous thrombin solution in alyophilization vessel is no greater than about 10 mm (such as, forexample, about 8 mm). In some such embodiments, a total lyophililzationtime is no greater than about 35 hours.

In some embodiments, the height of the aqueous thrombin solution in alyophilization vessel is no greater than about 20 mm (such as about15-19 mm, for example about 17 mm). In some such embodiments, a totallyophililzation time is no greater than about 68 hours.

In some embodiments, lyophilization comprises: i) subjecting the aqueousthrombin solution to a freezing procedure to produce a frozen proteinsolution; ii) subjecting the frozen thrombin solution of step i) to aprimary drying procedure; and iii) subjecting the product of step ii) toa secondary drying procedure.

In some embodiments, the freezing procedure is carried out at a freezingtemperature of about −45° C. to about −55° C.

In some embodiments, lyophilization is carried out on a lyophilizationshelf and the freezing procedure comprises maintaining the lyophilizershelf at a freezing temperature of about −45° C. to about −55° C., suchas, for example, about −50° C.

In some embodiments, the freezing procedure is carried out at a pressureof about 1 atmosphere.

In some embodiments, the height of the aqueous thrombin solution in alyophilization vessel is no greater than about 10 mm (such as, forexample about 8 mm). In some such embodiments, the freezing temperatureis maintained for no longer than about 5 hours.

In some embodiments, the height of the aqueous thrombin solution in alyophilization vessel is no greater than about 20 mm (such as, 15-19 mm,for example about 17 mm). In some such embodiments, the freezingtemperature is maintained for no longer than about 6 hours.

In some embodiments, the temperature of the lyophilizer shelf is broughtto the freezing temperature over a period of no greater than about 2.5hours.

In some embodiments, the primary drying procedure comprises maintainingthe lyophilizer shelf at a primary drying temperature of about −12° C.to about −18° C. (such as, for example, about −15° C.) at a pressure ofabout 100 μBar to about 160 μBar.

In some embodiments, the temperature of the lyophilizer shelf is broughtto the primary drying temperature over a period (also referred to hereinas a primary drying ramp) of about 80 to about 90 minutes.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 10 mm (such as, forexample, about 8 mm), the primary drying temperature and pressure aremaintained for no longer than about 13 hours (such as, for example,about 11 to about 13 hours).

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 20 mm (such as about15 to about 19 mm, for example, about 17 mm), the primary dryingtemperature and pressure are maintained for no longer than about 31hours.

In some embodiments, the secondary drying procedure comprisesmaintaining the lyophilizer shelf of step ii) at a secondary dryingtemperature of about 20° C. to about 30° C. at a pressure of less thanabout 50 μBar (such as, for example, less than about 20 μBar).

In some embodiments, the temperature of the lyophilizer shelf is broughtto the secondary drying temperature over a period of about 60 to about90 minutes

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 10 mm (such as, forexample, about 8 mm), the secondary drying temperature and pressure aremaintained for no longer than about 11 hours (such as, for example,about 9.5 to about 11 hours).

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 20 mm (such as,about 15 to about 19 mm, for example, about 17 mm), the secondary dryingtemperature and pressure are maintained for no longer than about 15hours (such as, for example, about 11 to about 13 hours). In some suchembodiments, prior to step iii), the lyophilizer shelf of step ii) ismaintained at an intermediate drying temperature of about 5 to about 15°C. (such as, for example, about 10° C.).

In some embodiments, the lyophilizer shelf of step ii) is maintained atthe intermediate drying temperature at a pressure of 120 μBar,

In some embodiments, the intermediate drying temperature is maintainedfor no longer than about 13 hours.

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 10 mm (such as, forexample, about 8 mm), lyophilization on a lyophilizer shelf comprises:

-   -   a) bringing the temperature of the lyophilizer shelf to a        freezing temperature of about −50° C., over a time period in the        range of about 1.5 to about 2.5 hours;    -   b) maintaining the freezing temperature of step a) for a time        period in the range of about 4 to about 6 hours to produce a        frozen thrombin solution;    -   c) increasing the temperature of the lyophilizer shelf of        step b) to a primary drying temperature of about −15° C. and        bringing the pressure to from about 100 to about 160 μBar, over        a time period in the range of about 50 to about 90 minutes;    -   d) maintaining the primary drying temperature and pressure for a        time period in the range of about 11 to about 13 hours;    -   e) increasing the temperature of the lyophilizer shelf of        step d) to a secondary drying temperature of about 25° C. and        decreasing the pressure to less than about 50 μBar, over a time        period in the range of about 60 to about 90 minutes; and    -   f) maintaining the secondary drying temperature and pressure for        a time period in the range of about 9.5 to about 11 hours,

In some embodiments, wherein the height of the aqueous thrombin solutionin a lyophilization vessel is no greater than about 20 mm (such as about15 to about 19, for example about 17 mm), lyophilization on alyophilizer shelf comprises:

-   -   a) bringing the temperature of the lyophilizer shelf to a        freezing temperature of about −50° C., over a time period in the        range of about 1.5 to about 2.5 hours;    -   b) maintaining the freezing temperature for a time period in the        range of about 4 to about 6 hours to produce a frozen thrombin        solution;    -   c) increasing the temperature of the lyophilizer shelf of        step b) to a primary drying temperature of about −15° C. and        decreasing the pressure to from about 100 to about 160 μBar,        over a time period in the range of about 30 to about 70 minutes;    -   d) maintaining the primary drying temperature and pressure for a        time period in the range of about 30 to about 32 hours;    -   e) increasing the temperature of the lyophilizer shelf of        step d) to a secondary drying temperature of about 25° C. and        decreasing the pressure to less than about 50 μBar, over a time        period in the range of about 40 to about 80 minutes; and    -   f) maintaining the secondary drying temperature and pressure for        a time period in the range of about 13 to about 17 hours.

In some embodiments, steps a) and b) are carried out at atmosphericpressure.

In some embodiments, there is provided a solid thrombin compositionobtainable according any of the methods described herein.

In some embodiments, the solid thrombin composition has a water contentof not more than about 3% (w/w) and a thrombin activity recovery of atleast 95%.

In some embodiments, the thrombin composition has a water content of notmore than about 1.5% (w/w) and a thrombin activity recovery of at least98%.

In some embodiments, the solid thrombin composition is stable for atleast 2 years under non-freezing storage conditions (such as at roomtemperature).

In some embodiments, the solid thrombin composition comprises about19.5% to about 78% (w/w of total composition) mannitol, about 1% toabout 20% (w/w of total composition) sodium acetate; about 2% to about53% (w/w of total composition) albumin; about 2.5% to about 31% (w/w oftotal composition) calcium chloride; and about 6% to about 45% (w/w oftotal composition) sodium chloride.

In some embodiments, the solid thrombin composition comprises about 22%to about 66% (w/w of total composition) mannitol, about 1.5% to about10% (w/w of total composition) sodium acetate; about 2.5% to about 43%(w/w of total composition) albumin; about 4% to about 17% (w/w of totalcomposition) calcium chloride; and about 9.5% to about 25% (w/w of totalcomposition) sodium chloride.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. In addition, the descriptions,materials, methods, and examples are illustrative only and not intendedto be limiting. Methods and materials similar or equivalent to thosedescribed herein can be used in the practices of the present invention.

As used herein, the term “cake” or “solid cake” refers to a porous andspongy structure-like composition resulting from the lyophilizationprocess.

As used herein, the term “collapse” with regard to a cake refers to thepoint at which the cake can no longer support its own structure.

As used herein, the term “solid composition” refers to a compositionhaving a water content of equal to or less than about 3% (w/w) watersuch as equal to or less than 1.5%, based on the total weight of thesolid composition.

As used herein, the term “thrombin activity recovery” refers to thethrombin activity in the solid composition upon reconstitution, ascompared to the initial thrombin activity in the aqueous thrombinsolution prior to lyophilization. Typically, the activity recovery ispresented in percentage.

As used herein, the term “stable” with regard to a lyophilized/solidthrombin composition is, for example, a composition that cansubstantially support its own structure without collapse of the cake,and/or a composition which retains at least 80% of its activity (such as90%, 95% or higher) for at least 2 years at non freezing temperaturestorage conditions e.g. at a temperature of 2-8° C. and up to roomtemperature such as less than 25° C. In one embodiment of the invention,the solid composition is stable for 2 years when stored at roomtemperature. Typically, collapse is most evidently characterized by theloss of cake structure during drying. Collapse usually results in astructure whose volume is significantly smaller than the volume of theformulation used to prepare the cake. Furthermore, a reduction of themean pore size and porosity as well as an increase in bulk density canbe observed.

The term “lyophilization” typically refers to the process of freezing asolution and then reducing the concentration of water e.g. bysublimation to levels which do not support biological or chemicalreactions. The resulting lyophilized composition may be stored for arelatively long period of time. Following storage, the lyophilizedcomposition can be used as a powder or can be reconstituted by theaddition of various volumes of an aqueous solution. The volume addedduring reconstitution can be similar to the volume of the solutionbefore lyophilization, lower or higher.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, integers, steps or components but do not preclude the additionof one or more additional features, integers, steps, components orgroups thereof. These terms encompass the terms “consisting of” and“consisting essentially of”.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

As used herein the term “about” refers to ±10%.

The solutions and methods described herein enable a solid thrombincomposition to be obtained by a shorter lyophilization cycle as comparedto lyophilization methods currently known in the art, thereby increasinglyophilization capacity and reducing production costs. Solid thrombincompositions obtained using the solutions and methods described hereinhave a low water content following lyophilization, as compared to knownthrombin compositions, are stable during storage at room temperatureover relatively long time periods, and show a high percentage ofthrombin activity recovery upon reconstitution.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are described herein with reference tothe accompanying figures. The description, together with the figures,makes apparent to a person having ordinary skill in the art how someembodiments of the invention may be practiced. The figures are for thepurpose of illustrative discussion and no attempt is made to showstructural details of an embodiment in more detail than is necessary fora fundamental understanding of the invention. For the sake of clarity,some objects depicted in the figures are not to scale.

In the Figures:

FIG. 1 shows the visual appearance of a cake obtained following anexemplary short lyophilization cycle as described herein.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

The invention, in some embodiments thereof, relates to methods forlyophilization of an aqueous thrombin solution, thrombin solutions foruse in such lyophilization methods, and solid thrombin compositionsproduced by such methods.

The principles, uses and implementations of the teachings herein may bebetter understood with reference to the accompanying description. Uponperusal of the description, one skilled in the art is able to implementthe invention without undue effort or experimentation.

Before explaining at least one embodiment in detail, it is to beunderstood that the invention is not necessarily limited in itsapplication to the details of construction and the arrangement of thecomponents and/or methods set forth in the following description. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. The phraseology and terminology employedherein are for descriptive purpose and should not be regarded aslimiting.

As mentioned above, known thrombin solutions vary considerably in thenumber and types of excipients present, and in the concentrations of theindividual excipients.

EXAMPLES

Materials and Methods

Thrombin Stock Solution:

The thrombin stock solution used for preparing the aqueous thrombinsolutions described in the Examples below comprised about 3,500 IU/mlthrombin, about 200 mM NaCl, with stabilizers 2% mannitol and 0.2% humanserum albumin (HSA) (unless indicated otherwise). The stock solution wasprepared from Factor II (pro-thrombin) of human plasma that wasactivated and purified by sequential use of diethylaminoethyl (DEAE)cellulose anion exchange resin and SP cation exchanger resin,essentially as described in U.S. Pat. No. 5,143,838, hereby incorporatedby reference.

Lyophilization:

A. Lyophilization of a Thrombin Solution of Height 8 mm within theLyophilization Vessel:

Lyophilization was carried out on a lyophilizer shelf of a ChristEpsilion 2-8D freeze drier (Christ, Germany) in 8 ml siliconized glassvials (Schott, Germany). Each vial was filled with 2 ml aqueous thrombinsolution. The height of the solution in each vial was approximately 8mm.

Two different short lyophilization cycles were carried out as specifiedin Table 1 (a total time of 34 hours) and Table 2 (a total time of 30hours) below. Temperatures given are those of the lyophilizer shelf.

Following lyophilization, a solid thrombin composition having a volumesimilar to the volume of the aqueous solution prior to lyophilizationwas obtained.

TABLE 1 Duration Pressure Temperature Step (hr:min) (μbar) (° C.)Freezing ramp  2 Atm RT to −50° C. Freezing soak  5 Atm −50° C. Primarydrying ramp 1:30 Atm to −50° C. to −15° C. 140 μBar Primary drying soak13 140 μBar −15° C. Secondary drying ramp 1:30 140 μBar to −15° C. to25° C. 12 μBar Secondary drying soak 11  12 μBar  25° C. Total Time 34

As used herein, the term “soak” with regard to a freezing or dryingprocess refers to maintaining the composition being lyophilized at aconstant temperature and pressure for a specified period of time inorder to effect freezing or drying, respectively.

As used herein, the term “ramp” with regard to a freezing or dryingprocess refers to a step in which the temperature and pressure of thecomposition being lyophilized is gradually changed over a specifiedperiod of time in order to bring the composition to a specified freezingor drying temperature and pressure respectively.

TABLE 2 Duration Pressure Temperature Step (hr:min) (μbar) (° C.)Freezing ramp  2 Atm RT to −50° C. Freezing soak 4:40 Atm −50° C.Primary drying ramp 1:20 Atm to −50° C. to −15° C. 140 μBar Primarydrying soak 11 140 μBar −15° C. Secondary drying ramp 1:30 140 μBar to−15° C. to 25° C. 12 μBar Secondary drying soak 9:30  12 μBar 25° C.Total Time 30B. Lyophilization of a Thrombin Solution of Height 17 mm within theLyophilization Vessel:

Lyophilization was carried out on a lyophilzer shelf of a ChristEpsilion 2-8D freeze drier (Christ, Germany) in LYOGUARD® cups (GORE®).Each vial was filled with 52 ml aqueous thrombin solution. The height ofthe solution in each vial was approximately 17 mm.

A short lyophilization cycles were carried out as specified in Table 3(a total time of 68 hours). Temperatures given are those of thelyophilizer shelf.

Following lyophilization, a solid thrombin composition having a volumesimilar to the volume of the aqueous solution prior to lyophilizationwas obtained.

TABLE 3 Duration Pressure Temperature Step (hr:min) (μbar) (° C.)Freezing ramp  2:00 Atm RT to −50° C. Freezing soak  5:40 Atm −50° C.Primary drying ramp  0:50 Atm to −50° C. to −15° C. 120 μBar Primarydrying 31:00 120 μBar −15° C. Intermediate drying  0:30 120 μBar −15° C.to 10° C. ramp Intermediate drying 12:00 120 μBar  10° C. Secondarydrying ramp  1:00 140 μBar to 10° C. to 25° C. 12 μBar Secondary drying15:00  12 μBar  25° C. Total Time 68:00Quantification of Water Content within the Lyophilized ThrombinComposition:

Water content determination was carried out using the volumetric KarlFischer Titration method (KFT), which is based on the US Pharmacopoeiaassay (USP 27, <921>, P. 2398-2399). Prior to the titration, the waterwas extracted from the lyophilized composition by adding dried methanolto the vial containing the lyophilized composition and shaking the vial.A sample from the supernatant were removed for the titration.

Determination of Thrombin Activity:

Thrombin activity of aqueous thrombin solutions was determined using aClotting Time assay by measuring thrombin clotting activity in thedifferent solutions according to the modified European PharmacopeiaAssay (0903/1997) procedure. Briefly, standard solutions of thrombin (4,6, 8 and 10 IU/ml) or the test solutions were incubated for 2 minutes at37° C. Then, 40 μl of each test solution or standard solution was mixedwith 160 μl fibrinogen solution (0.1%; Enzyme research; cat No FIB12800L) and clotting time was measured. A calibration curve of logclotting times vs. log thrombin concentration was plotted using thestandards. Thrombin activity in the different test solutions wasdetermined by the clotting time obtained (calculated automatically by aclotting machine (Diagnostica Stago STart Coagulation Analyzer)interpolated from the calibration curve and multiplied by the dilutionfactor).

Thrombin Activity Recovery Following Reconstitution (% of InitialActivity):

Thrombin activity was measured as described above in the test solutionsprior to lyophilization, and in the solid thrombin composition obtainedby lyophilization, after reconstitution with purified water to theoriginal volume. The recovered activity was calculated by dividing theactivity obtained in the solid thrombin composition followingreconstitution by the activity obtained in the thrombin solution beforelyophilization and multiplying by 100.

Qualitative and Quantitative Evaluation of Solid Thrombin CompositionsObtained by a Short Lyophilization Process:

Solid thrombin compositions obtained by subjecting different aqueousthrombin solutions to a short lyophilization cycle were evaluated by thefollowing parameters: water content of the solid compositions, thrombinactivity recovery following reconstitution of the solid compositions,and structural appearance of the cake (by visual inspection). Typically,a cake having a “good structural appearance” is defined as a cake havinga volume similar to that of the aqueous thrombin solution prior tolyophilization, is a monolithic block, has uniform porosity throughoutthe solid composition, and having no apparent wet areas.

For determining the effect of a short lyophilization process ondifferent thrombin formulations, several aqueous thrombin solutionscomprising different ingredients, e.g. different saccharides, salts, andexcipients at different concentrations were prepared from the thrombinstock solution described in the “MATERIALS AND METHODS” section, and thesolutions were then lyophilized using the short lyophilization cycle, asdetailed in Tables 1, 2 or 3.

Example 1 Use of Different Saccharides and Concentrations or a SugarAlcohol in a Thrombin Solution Subjected to a Short Lyophilization Cycle

Disaccharides (sucrose and maltose), and a sugar alcohol (mannitol) atdifferent concentrations were used in the preparation of aqueousthrombin solutions, and solid thrombin compositions obtained by use ofsuch thrombin solutions in a short lyophilization cycle were studied.

Mannitol was tested at concentrations of 1.6%, 2.1%, 2.6% and 4.6% (w/v)by addition of mannitol to the diluted stock solution as describedbelow; and sucrose and maltose were tested at a concentration of 2%(w/v). [For preparing the thrombin test solutions, the thrombin stocksolution described above was diluted 1:3.5 with a solution providing thefollowing final compositions: 0.6% human serum albumin, 20 mM sodiumacetate (0.27%), 130 mM NaCl (0.76%) and 0.6% CaCl₂ in water at pH 7.0,and the tested saccharide or sugar alcohol was added to the solution inthe concentrations listed above. In the case of mannitol, the solutionwas supplemented to the listed concentration (following dilution of thestock, the solution comprised about 0.6% mannitol).

Of note, human serum albumin present in the thrombin stock solution wasalso present in all test solutions as a “background ingredient” in aconcentration of approx. 0.06%. Also, the stock solution comprised 0.6%mannitol.

The prepared aqueous thrombin solutions were lyophilized using the shortlyophilization cycle as described in Table 2 above, to obtain solidthrombin compositions. The water content of each solid composition, andthrombin activity recovery following reconstitution of the lyophilizedcomposition were measured. The results are presented in Table 4 below.

TABLE 4 Water content in the Thrombin Saccharide/sugar alcohol solidcomposition Activity Recovery Tested (%) (%) Sucrose 2% 0.4 96 Maltose2% 0.6 95 Mannitol 1.6% 0.6 100 Mannitol 2.1% ND 100 Mannitol 2.6% 0.698 Mannitol 4.6% 1.7 100

The results showed that each of the tested saccharides and sugaralcohols, when present in the aqueous thrombin solution at aconcentration of 2.6% or lower, resulted in solid thrombin compositionshaving a low water content (0.4-0.6%) following a short lyophiliztioncycle, and high thrombin activity recovery (95-100%) followingreconstitution of the solid composition.

The results also showed that the aqueous thrombin solutions resulted incakes that maintained their structure without collapse, and which had agood structural appearance as defined above.

In contrast, an aqueous thrombin solution comprising 4.6% mannitol, whensubjected to a short lyophilization cycle, resulted in a shrunken cakewith a higher water content of 1.7%.

These results show that an aqueous thrombin solution for use in a shortlyophilization cycle advantageously comprises about 1.6% to less thanabout 4.6% saccharides or sugar alcohols in order to obtain a solid andstable (structurally and functionally) thrombin composition.

Example 2 Use of Different Concentrations of Human Serum Albumin in aThrombin Solution Subjected to a Short Lyophilization Cycle

Different concentrations of HSA were used in the preparation of aqueousthrombin solutions, and solid thrombin compositions obtained by use ofsuch thrombin solutions in a short lyophilization cycle were studied.

HSA was tested at the following concentrations: 0.2%, 0.6%, 3% and 10%.

The solutions were prepared from the thrombin stock solution by 1:3.5dilution with a solution providing the following final composition: 2.6%mannitol, 20 mM sodium acetate (0.27%), 130 mM NaCl (0.76%) and 0.6%CaCl₂ in water at pH 7.0.

Of note, human serum albumin was present in all test solutions at aconcentration of approx. 0.06%, (in addition to the added HSAconcentrations listed above).

The prepared aqueous thrombin solutions were lyophilized using the shortlyophilization cycle described in Table 2 above, wherein the solutionhad a height of about 8 mm in the lyophilization vessel, to obtain solidthrombin compositions. The water content of each solid composition, andthrombin activity recovery following reconstitution of the solidcomposition were measured. The results are presented in Table 5 below.

TABLE 5 Water content in the solid composition Thrombin activityrecovery HSA concentration (%) (%) (%) 0.2 3.0 100 0.6 0.6 98 3 ND 89 100.3 28

The results showed that changing the HSA concentration had a significanteffect on the water content in the solid thrombin composition and on thethrombin activity recovery following reconstitution of the solidcomposition.

More specifically, it was shown that a thrombin solution containing 0.6%HSA resulted in a solid composition having both relatively low watercontent and a high recovery of thrombin activity upon reconstitution. Incontrast, thrombin solutions with lower HSA concentrations resulted insolid compositions with increased water content, while solutions withhigher HSA concentrations resulted in a lower recovery of thrombinactivity upon reconstitution.

Therefore, it is shown that optimal thrombin solutions advantagouslycomprise HSA at a concentration of higher than about 0.2% to lower thanabout 3%.

Example 3 Use of Different Salts and Concentrations in a ThrombinSolution Subjected to a Short Lyophilization Cycle

A. Effect of Sodium Chloride (NaCl) Concentration:

Different concentrations of NaCl (90 mM, 120 mM and 150 mM, i.e. 0.5%w/v, 0.7 w/v, and 0.9 w/v, respectively) were used in the preparation ofaqueous solutions devoid of thrombin, and solid compositions obtained byuse of such solutions in a short lyophilization cycle were studied,wherein the height of the solutions in a lyophilization vessel was about8 mm.

The solutions contained, in addition to the different concentrations ofNaCl, 2% mannitol, 0.6% HSA, 20 mM sodium acetate (0.27%), and 0.6%CaCl₂ at pH 7.0.

The solutions were lyophilized using a short cycle similar to thatdescribed in Table 2 above, wherein the height of the solution in thelyophilization vessel was about 8 mm, wherein the primary drying soakwas an hour longer than described in Table 2.

Following lyophilization, the water content in the solid composition wastested. The results are presented in Table 6 below.

TABLE 6 Water content in the solid composition NaCl Concentration (%) 90 mM/0.5% w/v   3% 120 mM/0.7% w/v 1.2% 150 mM/0.9% w/v <1.2% 

As shown in the Table, the water content was inversely proportional toNaCl concentration up to 150 mM.

In additional similar experiments using thrombin solutions and solutionsdevoid of thrombin (data not shown), no further decrease in watercontent was found when NaCl concentration was increased beyond 150 mM.

NaCl concentrations of 300 mM (1.75% w/v) and above had an inhibitoryeffect on thrombin activity as measured by clotting time assay (data notshown).

With regards to structural appearance of the solid composition, a cakeprepared from a solution comprising 90 mM NaCl had a poor appearance,being shrunken compared to the volume of the solution from which it wasprepared, and having a granular consistency, with non-uniform porositythroughout the solid composition.

Therefore, it was concluded that advantageously optimal thrombinsolutions comprise NaCl at a concentration of at least 120 mM (0.7% w/v)to lower than 300 mM (1.75% w/v).

B. Effect of Partial Replacement of NaCl by Potassium Chloride (KCl)

The potential use of KCl instead of NaCl in the thrombin solution wasassessed. Since NaCl is present in the thrombin stock solution used toprepare the test solutions, it cannot be completely removed and thus thesolution was supplemented with 65 mM KCl (bringing the solution to afinal salt concentration of about 125 mM). Also, an additional thrombinsolution was tested, comprising the stock solution supplemented withNaCl, bringing the solution to a final salt concentration of 130 mM).

Solutions supplemented with NaCl or with KCl were prepared by dilutingstock solution 1:3.5, to provide a NaCl concentration of 60 mM. A firstsolution was then supplemented to an NaCl concentration of 130 mM; and asecond solution was supplemented with 65 mM KCl.

The stock solution was diluted so as to provide the following finalcomposition: 2.6% mannitol, 20 mM sodium acetate (0.27%), and 0.6% CaCl₂in water at pH 7.0.

The prepared solutions were lyophilized using the short lyophilizationcycle described in Table 2 above, wherein the solution has a height inthe lyophilization vessel of about 8 mm, and the water content in thesolid compositions and the thrombin activity recovery followingreconstitution of the solid composition were measured. The results arepresented in Table 7 below.

TABLE 7 Water content in the Thrombin activity Salt Tested solidcomposition (%) recovery (%) 130 mM NaCl (0.76% w/v) 0.6 98 65 mM (0.49%w/v) KCl; and 5.2 100 60 mM (0.35% w/v) NaCl

It was found that partial replacement of NaCl with KCl in the solutionhad a detrimental effect on the water content of the solid thrombincomposition obtained following a short lyophilization cycle (5.2% watercontent of solid composition following lyophilization of a solutioncontaining NaCl plus KCl, as compared to 0.6% following lyophliztion ofa solution containing NaCl without KCl). Similar thrombin activityrecovery was obtained following reconstitution of a solid compositionobtained from lyophilization of a solution comprising 130 mM NaCl and asolution comprising 65 mM KCl and 60 mM NaCl, as shown in Table 7.

Visual inspection of the structural appearance of the solid compositionrevealed that replacement of NaCl with KCl resulted in a shrunken cake.

It was therefore concluded that advatagously in order to obtain a solidcomposition with a relatively low water content, it is preferred thatKCl is absent from the thrombin solution.

It is further concluded that an optimal aqueous thrombin solution thatyields a solid composition with low water content and a high thrombinactivity recovery following lyophilization using a short cycle comprisesabout 1.6% to less than about 4.6% of saccharide or sugar alcohol, atleast about 120 mM (0.7% w/v) to lower than about 300 mM (1.75% w/v)sodium chloride, and higher than about 0.2% to lower than about 3%albumin, in addition to about 0.3 to about 1.2% calcium chloride; andabout 0.14 to about 0.7% sodium acetate.

Example 4 Use of the Solutions Described Herein and Solutions Similar toPrior Art Solutions in a Short Lyophilization Cycle with Height ofSolution in Lyophilization Vessel of 8 mm

Three solutions were prepared: a solution as described herein, and twosolution which are similar to prior art solutions, as follows:

-   -   1. A thrombin solution as described herein, comprising 2%        mannitol, 0.6% human serum albumin, 20 mM sodium acetate        (0.27%), 130 mM sodium chloride (0.76%) and 40 mM CaCl₂ (0.6%)        in distilled water at pH 7.0.    -   2. A thrombin solution similar to that disclosed in EP 1766003B1        (Jiang et al.), comprising 3% sucrose, 4% mannitol, 150 mM NaCl        (0.9%), 0.1% polyethylene glycol 3350 (PEG3350), 4 mM CaCl₂        (0.04%); and 5 mM (0.08%) histidine in distilled water at pH        6.0.    -   3. A thrombin solution similar to that disclosed in EP 813598B1        (MacGregor et al.), comprising 40 mM (0.78%) gluconic acid, 20        mM (0.5%) tri-sodium citrate and 150 mM NaCl (0.9%) in distilled        water at pH 6.5.        The components of the solutions are further presented in Table        8.

TABLE 8 Solution 1 Solution 2 Solution 3 Mannitol   2%   4% — Albumin 0.6% — — Sodium acetate 0.27% — — Sodium chloride 0.76% 0.9% 0.9%Calcium chloride  0.6% 4 mM (0.04%) — Sucrose —   3% — PEG — 0.1% —Histidine — 5 mM (0.08%) — Gluconic acid — — 40 mM (0.78%) Tri-sodiumcitrate — — 20 mM (0.5%) 

All three thrombin solutions were prepared by diluting the thrombinstock solution described above 1:3.5 with a solution comprising theingredients listed in each solution 1-3. Mannitol and human serumalbumin were present in all three test solutions in a concentration ofapprox. 0.6% and 0.06%, respectively, as background ingredients

The solutions were lyophilized using the short lyophilization cyclesdescribed in Table 1 or Table 2 above (wherein the height of thesolution in the lyophilization vessel was about 8 mm) Water content ofthe solid thrombin composition, and thrombin activity recovery afterreconstitution of the solid thrombin composition were measured, asdescribed above.

FIG. 1 shows the visual appearance of a solid thrombin compositionobtained following a short lyophilization of the aqueous thrombinsolution described herein (solution 1; #1 in FIG. 1); and a solidthrombin composition obtained following a short lyophilization of asolution according to EP 1766003B1 (solution 2; #2 in FIG. 1). The solidcomposition shown in FIG. 1 was obtained using the cycle as described inTable 1. Similar results were obtained when the cycle as described inTable 2 was used.

Visual inspection of the appearance of the solid thrombin compositionsrevealed that the solid compositions obtained following lyophilizationof the thrombin solution according to the invention and of solution 3had a “good structural appearance” according to the definition discussedabove. However, the cake obtained following use of the thrombin solutionaccording to EP 1766003B1 (solution 2) in the short lyophilization cyclehad a granulated structure.

Water content in the three solid thrombin compositions and the thrombinactivity recovery of the solid compositions is presented in Table 9below.

TABLE 9 Thrombin Water Content in the activity Average solid composition(%) recovery (%) throm- Throm- Cycle Cycle Average Cycle Cycle bin binof of water of of activity Solution Table Table content Table Tablerecovery Tested 1 2 (%) ±SD 1 2 (%) ±SD Solution 0.8 0.6  0.7 ± 0.14 10098 99 ± 1.4 1 Solution 3.2 4.1 3.65 ± 0.64 92 96 94 ± 2.8 2 Solution 1.63.3 2.45 ± 1.2  93 91 92 ± 1.4 3

As shown in Table 9, it was found that following the shortlyophilization cycle (as described in Tables 1 or 2), the lowest averagewater content was found in the lyophilized solid composition preparedusing the solution described herein (0.7±0.14%).

As further shown in Table 9, it was found that following reconstitutionof the short-cycle lyophilized solid compositions, the highest thrombinactivity recovery was obtained in the solid compositions prepared usingthe solution described herein (99±1.4%) according to Tables 1 or 2.

Example 5 Use of the Solutions Described Herein in a ShortLyophilization Cycle with Height of Solution in Lyophilization Vessel of17 mm

A first thrombin solution as described herein, comprising 2% mannitol,0.6% human serum albumin, 20 mM sodium acetate (0.27%), 130 mM sodiumchloride (0.76%) and 40 mM CaCl₂ (0.6%) in distilled water at pH 7.0;and a second solution, similar to the first solution, but comprising asodium chloride concentration of lower than 120 mM (0.7%) sodiumchloride (lower than 0.76%) were lyophilized. The first solution waslyophilized using the short lyophilization cycles described in Table 3above; and the second solution (with the lower sodium chlorideconcentration) was lyophilized in a lyophilizatin cycle having aduration of 108 hours (wherein the height of each solution in thelyophilization vessel was about 17 mm).

Water content of both solid thrombin compositions, and thrombin activityrecovery after reconstitution of both solid thrombin compositions weremeasured, as described above.

In both solid compositions, water content was 1.5% and the thrombinactivity recovery was 100%.

The results show that the thrombin solution according to the inventionenabled to shorten the duration of the lyophilization cycle by about37%. In conclusion, the thrombin solution described hereinadvantageously enables use of a short lyophilization cycle that yields asolid composition having low water content and the high thrombinactivity recovery as compared to known thrombin solutions.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the scope of the appendedclaims.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the invention.

The invention claimed is:
 1. A solid composition comprising thrombin,obtained by lyophilization of an aqueous thrombin solution, the methodcomprising: providing the aqueous thrombin solution comprising about 1to less than about 4.6% w/v of a single saccharide or sugar alcohol; atleast 0.7 to lower than about 1.75% w/v sodium chloride; about 0.2 toabout 3% w/v albumin; calcium chloride; and sodium acetate, wherein theaqueous thrombin solution is devoid of polyethylene glycol; andlyophilizing the aqueous thrombin solution, wherein a height of theaqueous thrombin solution in a lyophilization vessel is in the range of6 mm to 10 mm, and wherein a total lyophilization time is no greaterthan about 35 hours; wherein the obtained solid composition is devoid ofpolyethylene glycol, comprises thrombin, a single saccharide or sugaralcohol, and sodium chloride, and wherein the solid composition has awater content of less than 3%, by total weight of the composition. 2.The solid thrombin composition of claim 1, wherein the aqueous thrombinsolution comprises about 200 to about 2000 IU/ml thrombin; about 0.3 toabout 1.5% w/v calcium chloride; and about 0.14 to about 1% w/v sodiumacetate.
 3. The solid thrombin composition of claim 2, wherein theaqueous thrombin solution comprises: about 1.6 to less than about 4.6%w/v single saccharide or sugar alcohol; about 0.7 to about 1.7% w/vsodium chloride; higher than about 0.2 to lower than about 3% albuminw/v; about 0.3 to about 1.2% w/v calcium chloride; and about 0.14 toabout 0.7% w/v sodium acetate.
 4. The solid thrombin composition ofclaim 3, wherein the single saccharide or sugar alcohol is present at aconcentration of 2% w/v; the albumin is present at a concentration ofabout 0.6% w/v; the sodium chloride is present at a concentration ofabout 0.76% w/v; the calcium chloride is present at a concentration ofabout 0.6% w/v and the sodium acetate is present at a concentration ofabout 0.27% w/v.
 5. The solid thrombin composition of claim 1, theaqueous thrombin solution consisting essentially of thrombin; singlesaccharide or sugar alcohol; sodium chloride; albumin; calcium chloride;and sodium acetate.
 6. The solid thrombin composition of claim 1,wherein the single sugar alcohol is mannitol.
 7. The solid thrombincomposition of claim 6, wherein the mannitol is present at aconcentration of about 2% w/v.
 8. The solid thrombin composition ofclaim 1, wherein the aqueous thrombin solution is devoid of histidine.9. The solid thrombin composition of claim 1, comprising sodium chlorideat an amount in the range of 6% to 45% by weight of the solidcomposition.